The present invention relates to a chromatographic method for the detection or analysis of polymorphisms in nucleic acids, and particularly to denaturing high performance liquid chromatography for such uses.
Approximately 4,000 human disorders are attributed to heritable genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the Human Genome Project is to find all genes associated with each disorder.
The most reliable diagnostic test for any specific genetic disease (or predisposition to a particular disease) is the identification of polymorphic variations in DNA sequence in affected cells that result in altered gene function and/or expression levels. In addition, certain polymorphic variations that are associated with predispositions for disorders, e.g., alleles that are associated with disease such as certain forms of cancer or Alzheimer""s disease, may allow prophylactic measure to be taken to help reduce or reverse the risk imposed by the polymorphic allele. Furthermore, responses to specific medications may depend on the presence of polymorphisms, making people with a particular polymorphism a better candidate for a medication than those not possessing the polymorphism. These and other reasons provide a great impetus for developing DNA or RNA screening as a practical tool for medical diagnostics.
Genetic polymorphisms and mutations can manifest themselves in several forms, such as point polymorphisms or point mutations where a single base is changed to one of the three other bases, deletions where one or more bases are removed from a nucleic acid sequence and the bases flanking the deleted sequence are directly linked to each other, insertions where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the overall sequence, and expansions and reductions of repeating sequence motifs. Large insertions and deletions, often the result of chromosomal recombination and rearrangement events, can lead to partial or complete loss of a gene. Of these forms of polymorphism, in general point polymorphisms are the most difficult to detect because they represent the smallest degree of molecular change.
The most definitive screening method to identify and determine polymorphisms such as SNPs in a nucleic acid requires determining the actual base sequence (Maxam and Gilbert, 1977; Sanger et al., 1977). Although such a method is the most accurate, it is also the most expensive and time consuming method. Restriction mapping analysis has some limited use in analyzing DNA for polymorphisms. If one is looking for a known polymorphism at a site which will change the recognition site for a restriction enzyme, it is possible simply to digest DNA with this restriction enzyme and analyze the relative sizes and numbers of fragments to determine the presence or absence of the polymorphism. (R. K. Saiki et al., Science 230 (1985), 1350-1354). This type of analysis is also useful for determining the presence or absence of gross insertions or deletions, but may not be useful in detecting smaller changes that do not result in a readily distinguishable change in restriction fragment size and/or number. Restriction mapping methods also generally require the use of hybridization techniques which are time consuming and costly.
The large-scale identification of single-nucleotide polymorphisms (SNPs) in the human as well as other model genomes such as yeast and Arabidopsis thaliana has been accomplished by methods such as fluorescence-based sequencing (P.-Y. Kwok, Q. et al., Genomics 31 (1996) 123-126), hybridization high-density variation-detection DNA chips (D. G. Wang et al., Science 280 (1998) 1077-1082; E. A. Winzeler et al., Science 281 (1998) 1194-1197), and high performance liquid chromatography (P. A. Underhill et al., Genome Res. 7 (1997) 996-1005; M. Giordano et al., Genomics, 56 (1999) 247-253; R. J. Cho et al., Nature Genet. 23 (1999) 203-207; and M. Cargill et al, Nature Genet. 22 (1999) 231-238). These and other methods have been used to identify thousands of SNPs. For this reason, the development of simple and inexpensive technology for the genotyping of SNPs of individuals (e.g., in a clinical setting) has become of great interest as the ability to discriminate between allelic forms of SNPs is increasingly seen as fundamental to future molecular genetic analysis of disease (N. Risch and K. Merikangas, Science 273 (1996) 1516-1517; F. S. Collins et al., Science 278 (1997) 1580-1581; L. Kruglyak, Nature Genet. 17 (1997) 21-24).
A number of additional methods are available for SNP genotyping such as allele-specific hybridization (R. K. Saiki et al., N. Engl. J. Med. 319 (1988) 537-541; M. Chee et al., Science 274 (1996) 610-614), nick translation PCR (L. G. Lee et al., Nucl. Acids Res. 21 (1993) 3761-3766; K. J. Livaket al., PCR Methods Appl. 4 (1995) 357-362), ligase chain reaction (D. Y. Wu and R. B. Wallace, Genomics 4 (1989) 560-560; D. A. Nickerson et al., Proc. Natl. Acad. Sci. USA 87 (1990) 8923-8927), allele-specific polymerase chain reaction (C. R. Newton et al, Nucl. Acids Res. 17 (1989) 2503-2516; D. Y. Wu et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2757-2760); Tm-shift genotyping (S. Germer and R. Higuchi, Genome Res. 9 (1999) 72-78), and minisequencing (A. Jalanko et al., Clin. Chem. 38 (1992) 39-43; P. Nyren et al., Anal. Biochem. 208 (1993) 171-175; T. T. Nikiforov et al., Nucl. Acids Res. 22 (1994) 4167-4175; T. Pastinen et al., Clin. Chem. 42 (1996) 1391-1397; G. S. Higgins et al., BioTechniques 23 (1997) 710-714; L. A. Haff and I. P Smirnov, Genome Res. 7 (1997) 378-388; C. A. Piggee et al., J. Chromatogr. A 781 (1997) 367-75; X. Chen et al., Genome Res. 9 (1999) 492-498; and B. Hoogendoom et al., Hum. Genet. 104 (1999) 89-93). The latter method, which is based on the annealing of a primer immediately upstream or downstream from the polymorphic site and its extension by one or more bases in the presence of the appropriate dNTPs and ddNTPs, has become very popular. It has been combined with a variety of techniques for detecting the extension products, including radiolabeling (A. Jalanko et al., Clin. Chem. 38 (1992) 39-43), luminous detection (P. Nyren et al, Anal. Biochem. 208 (1993) 171-175), colorimetric ELISA (T. T. Nikiforov et al., Nucl. Acids Res. 22 (1994) 4167-4175), gel-based fluorescent detection (T. Pastinen et al., Clin. Chem. 42 (1996) 1391-1397), mass spectrometry (G. S. Higgins et al., BioTechniques 23 (1997) 710-714; L. A. Haff and I. P Smimov, Genome Res. 7 (1997) 378-388), capillary electrophoresis (C. A. Piggee et al., J. Chromatogr. A 781 (1997) 367-75), fluorescence polarization (X. Chen et al., Genome Res. 9 (1999) 492-498), and most recently high-performance liquid chromatography (B. Hoogendoom et al., Hum. Genet. 104 (1999) 89-93).
All of the aforementioned genotyping techniques use the polymerase chain reaction as the initial sample pretreatment step. Many of these techniques thus require at least a two-step process to determine the presence of an SNP. Although some of the methods can be done in a single step in a single tube, these techniques require expensive fluorescent dye-labeled oligonucleotide probes (L. G. Lee et al., Nucl. Acids Res. 21 (1993) 3761-3766.; K. J. Livak et al., PCR Methods Appl. 4 (1995) 357-362). Others require additional steps such as hybridization or primer extension. Primer extension also requires prior purification of the PCR product from unincorporated dNTPs and oligonucleotides by either solid-phase extraction or enzymatic treatment with Shrimp Alkaline Phosphatase and Exonuclease I. For these reasons, genotyping is still a far more costly undertaking than identifying the presence of an SNP in the genome. This constitutes a severe limitation in the application of SNPs to genetic studies in the clinic and laboratories.
High-performance liquid chromatography (HPLC) has been used to identify and analyze polymorphisms in DNA, for example by detecting the presence of heteroduplices in DNA samples from an individual. The importance of preconditioning DNA prior to its contact with the column matrix had been recognized for the successful resolution of homo- and heteroduplex species under partially denaturing conditions, as it proved impossible to detect heteroduplices when the DNA sample was injected directly into the column without such preconditioning. (A. Hayward-Lester et al., in: F. Ferrxc3xa9 (Ed.), Gene Quantification, Birkhxc3xa4user Verlag, 1997, pp. 44-77; U.S. Pat. No. 5,795,976). Although techniques such as HPLC under partially denaturing conditions are powerful for identifying poymorphisms and detecting polymorphisms in the presence of a reference nucleic acid (i.e., by the formation of a homo- or heteroduplex with the reference nucleic acid), single nucleotide changes in an allele could not be directly determined using these techniques, even under optimum conditions. (See e.g., C. G. Huber et al., Anal. Biochem. 212 (1993) 351-358).
All of the methods in use today capable of screening broadly for genetic polymorphisms suffer from technical complications and are labor and time intensive. There is a need for new methods that can provide cost effective and expeditious means for screening genetic material.
The present invention provides a method for detecting polymorphisms in a nucleic acid, e.g., DNA or RNA, by 1) preconditioning a sample of nucleic acids to completely denature the nucleic acids, e.g., via heating and/or chemical treatment; and 2) performing high-performance liquid chromatography (HPLC) on the sample under denaturing conditions to identify the polymorphism of the nucleic acid. The nucleic acids to be analyzed are completely denatured prior to application of the sample to a stationary reverse-phase support and throughout the HPLC process. The sample mixture is eluted with a mobile phase containing an ion-pairing reagent and an organic solvent. Sample elution is also carried out under completely denaturing conditions.
The nucleic acid sample to be analyzed is generally injected and pre-mixed with the mobile phase prior to elution on the solid support. The sample is preferably injected into a pre-conditioned mobile phase, though it can also be passed through a xe2x80x9cpreconditioningxe2x80x9d tubing or pre-column placed between injector and column. This allows the sample to equilibrate before contact with the solid support, and provides a means for denaturation of the sample, e.g., by heating of the mobile phase-sample mixture or by contact of the sample with the alkaline environment of the mobile phase.
The stationary phase used in the present methods may be any reverse phase solid support, including monolith stationary phases and stationary phases based on particles. Reverse phase columns or column packing materials for use in the invention are typically composed of alkylated polymeric solid support materials such as silica, cellulose and cellulose derivatives such as carboxymethylcellulose, alumina, zirconia, polystyrene, polyacrylamide,polymethylmethacrylate, and styrene copolymers. In a preferred embodiment, the polymeric base material is a styrene-divinyl copolymer. Typically, the stationary support is composed of beads from about 1-100 microns in size.
The mobile phase contains an ion-pairing agent and an organic solvent. Ion-pairing agents for use in the method include lower primary, secondary and tertiary amines, lower trialkylammonium salts such as triethylammonium acetate and lower quaternary ammonium salts. A preferred tertiary amine is triethyl amine. Typically, the ion-pairing reagent is present at a concentration between about 0.05 and 1.0 molar. Organic solvents for use in the method include solvents such as methanol, ethanol, 2-propanol, acetonitrile, and ethyl acetate.
In one embodiment, the method of the invention utilizes thermal means to provide and maintain completely denaturing conditions of the mobile phase and the stationary phase during HPLC. When denaturation of the sample is provided by heating, preferably the apparatus used in performing the HPLC, e.g., the sample loop, preconditioning coil, and the column, are all maintained at a sufficient temperature to maintain denaturation of the nucleic acid in the sample.
In another embodiment of the invention, completely denaturing conditions are achieved and maintained by the presence of a compound that increases the pH of the mobile phase, e.g. NaOH. Sample elution is then carried out under pH conditions effective to maintain complete denaturation of the nucleic acids. In such cases, a lower column temperature (less than about 65xc2x0 C.) may be sufficient for determining polymorphisms in the sample.
In one particularly preferred embodiment of the present method, analysis of the nucleotide sequence of an oligomer is determined by applying a sample containing an oligomer to a C-18 alkylated polystyrene-divinylbenzene copolymer stationary support and eluting the mixture with a mobile phase containing triethylammonium acetate as the ion-pairing reagent and acetonitrile as the organic solvent at a temperature between about 70xc2x0-80xc2x0 C.
An advantage of the present invention is that the majority of possible transitions and transversions can be typed accurately.
Another advantage of the invention is that the method of the present invention can be used in conjunction with other methods of detecting and analyzing polymorphisms, e.g., detection by means of HPLC based heteroduplex detection under partially denaturing conditions and analysis using methods such as mass spectrometry.
The invention also provides a method for direct discrimination of alleles using completely denaturing HPLC. A DNA oligomer (e.g., an amplicon produced from a genetic region containing a known SNP) is amplified from the individual to be analyzed and the selected polymorphic site contained therein is identified using the separation method of the present invention. The polymorphism is detected by the sequence of the oligomer, and thus does not require the use of a reference oligomer to determine the presence of the polymorphism.
Isolation may be accomplished through any number of methods, including but not limited to amplification (e.g., PCR) or reverse transcription, and restriction digestion and purification. HPLC is performed using a reverse phase column. Such methods provide a fast, efficient and inexpensive method of direct allelic discrimination which does not require a positive control to identify single base polymorphisms.
It is an object of the present invention to provide methods for allelic discrimination using direct detection of nucleotide differences by HPLC analysis of PCR-generated amplicons.
It is an advantage of the present method that the oligomers may be rapidly genotyped without the need of a reference chromosome.
It is an advantage of the present method that the oligomers to be analyzed may be isolated using any number of different methods, including reverse transcription and PCR.
The invention also provides methods to diagnose and/or determine prognosis and appropriate treatment methods for a subject using the methods of the invention. The present methods of identifying polymorphisms can be used to identity nucleotide changes associated with a disease state, with a predisposition for a disease, with a particular prognosis, or with response to a particular therapeutic treatment.
It is yet another object of the present invention to detect polymorphisms to be used as genetic markers and/or diagnostic tools. This includes polymorphisms in regions of either high or low mutation, including polymorphisms in regions known to have great genetic variability across a population, mutations that are causative of a disorder. The present methods can also be used to detect very rare somatic mutations.
In another embodiment, a selected set of one or more single nucleotide polymorphisms is determined for a given group or population, and the information stored in a data storage computer system, e.g., a relational database system.
The invention also provides the production of polymorphism databases produced using the present methods. Such databases may be produced for any number of purposes such as forensic identification of an individual, linkage analysis, population studies, epidemiological surveys, and the like.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the protocols as more fully described below.